Center for Biologics, Evaluation and Research, U.S. Food and Drug Administration, Bethesda, MD 20892, United States.
J Virol Methods. 2009 May;157(2):133-40. doi: 10.1016/j.jviromet.2009.01.002. Epub 2009 Jan 30.
PCR-based reverse transcriptase (RT) assays are highly sensitive for broad detection of retroviruses. These assays are currently used for demonstrating the absence of retroviral contamination in vaccines and can also be applied to clinical and laboratory research to investigate low-virus replication. A single-tube fluorescent product-enhanced reverse transcriptase assay (STF-PERT) has been published that was highly sensitive for retrovirus detection (<10 virions), with enhanced reproducibility and increased efficiency [Sears, J.F., Khan, A.S., 2003. Single-tube fluorescent product-enhanced reverse transcriptase assay with AmpliWax (STF-PERT) for retrovirus quantitation. J. Virol. Meth. 108, 139-142]. In this report, the step-by-step setup and performance of the STF-PERT assay is described and sensitivity, reproducibility and specificity of the assay reported using three different RTs as standards: avian myeloblastosis virus (AMV) RT, murine leukemia virus (MMLV) RT, and human immunodeficiency virus type 1 (HIV-1) RT. Evaluation of virus stocks showed about 1-2 logs difference in RT detection and retrovirus quantitation with the different RT enzyme standards; in general, virus determination using HIV-1 RT was comparable to using the relevant virus RT.
基于聚合酶链反应（PCR）的逆转录酶（RT）检测法对广泛检测逆转录病毒具有高度敏感性。这些检测法目前用于证明疫苗中不存在逆转录病毒污染，也可应用于临床和实验室研究，以调查低病毒复制情况。已经公布了一种单管荧光产物增强逆转录酶检测法（STF-PERT），该方法对逆转录病毒检测具有高度敏感性（<10 个病毒粒子），具有增强的可重复性和更高的效率[ Sears，J.F.，Khan，A.S.，2003. 用于逆转录病毒定量的单管荧光产物增强逆转录酶检测法（STF-PERT）。J. Virol. Meth. 108，139-142]。在本报告中，描述了 STF-PERT 检测法的逐步设置和性能，以及使用三种不同的 RT 作为标准品报告的检测法的敏感性、重现性和特异性：禽髓细胞瘤病毒（AMV）RT、鼠白血病病毒（MMLV）RT 和人类免疫缺陷病毒 1 型（HIV-1）RT。对病毒库存的评估表明，使用不同的 RT 酶标准品进行 RT 检测和逆转录病毒定量的差异约为 1-2 个对数级；通常，使用 HIV-1 RT 进行病毒测定与使用相关病毒 RT 进行病毒测定相当。
